Abstract
The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.
Highlights
Adenylyl transferases (ATases) utilize adenosine triphosphate (ATP) to covalently modify proteins, nucleic acids, or small molecules with adenosine monophosphate (AMP), a reaction known as adenylylation or AMPylation
Mutational and bioinformatics analysis indicated that Fic proteins containing a strictly conserved HxFx(D/E)GNGRxxR signature motif in the active center typically display adenylylation activity [1,2,3,4,5], while Fic proteins with an active center deviating from this consensus are considered to have adopted different activities
We recently demonstrated that the Fic protein VbhT from Bartonella schoenbuchensis causes bacterial growth arrest when overexpressed in Bartonella or E. coli and that this effect can be repressed by co-expression with the anti-toxin VbhA, a small protein encoded upstream of VbhT [8]
Summary
Adenylyl transferases (ATases) utilize adenosine triphosphate (ATP) to covalently modify proteins, nucleic acids, or small molecules with adenosine monophosphate (AMP), a reaction known as adenylylation or AMPylation. The bacterial T3SS effector protein VopS from Vibrio parahaemolyticus and the surface antigen IbpA from Histophilus somni covalently attach the bulky AMP moiety onto a specific threonine or tyrosine, respectively, of the switch I region of Rho family GTPases [1,2]. This abrogates binding of downstream effectors and results in actin cytoskeleton collapse and concomitant cell detachment and death. The hosttargeted effector protein AnkX of Legionella pneumophila exhibiting an HxFxDANGRxxV signature motif displays phosphocholination activity towards the GTPase Rab1 [6]
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